DESCRIPTION: The hypotricous ciliates are remarkable organisms. Like all ciliates they have two nuclei; a transcriptionally silent micronucleus and a transcriptionally active macronucleus (nuclei). The DNA of the micronucleus is transmitted sexually to the next generation and in mitotic cell divisions by standard eukaryotic mechanisms. The DNA of the macronucleus is found in gene-sized pieces that amplified, is not transmitted sexually to the next generation, only a fraction of the micronuclear DNA (`10%) is present and the gene-sized pieces are segregated amitotically in the mitotic cell divisions. In addition, the nuclear genome uses TAA and TAG, which are normally stop codons, as glutamine codons. After conjugation, the DNA destined to become the macronucleus is amplified in polytene chromosomes. There is chromosome breakage, loss of large portions of the DNA and new telomeres are made and added on to the gene-sized pieces that are left. The sequences that are lost are referred to as internally eliminated sequences or IESs. So this nucleus is genetically a dead-end. It will not be transmitted to the next generation. IESs fall both in genes and between genes. Especially in the many examples of IESs that fall in the coding region, the excision of the IES must be exact. The starting point for these studies was the locus designated 81. It has five short IESs and three TBE1 elements. The TBE1 element is a 4.1 kb transposon that is present in the micronuclear genome in about 2,200 copies per haploid genome. All of these sequences are eliminated in the production of the macronucleus. They are flanked by AAT target duplications. There are also short IESs and there may be 105 short IESs in the genome. TBE1 has five long open reading frames. At least two of these are conserved in the four TBE1 elements that have been sequenced. The 57 kD ORF shows homology to protein kinases and zinc fingers and the 42 kD ORF shows homology to transposases, which include mariner. TBE2 and TBE3 have also been sequenced. They share about 40% sequence identity. When TBE1 excises, it does not appear to use gap-repair and it generates circular molecules. The RNA for the TBE1 elements is very labile, heterogeneous, and does not appear to be polyadenylated. Nested PCR and probing gave a signal, so there may a small fraction that is polyadenylated. Bulk PCR sequencing using a set of primers suggest that the 1,500 to 3,000 TBE1 have both primers and they are at a fairly canonical distance apart. Dr. Herrick's proposal seeks to understand how these the sequence similarity is maintained among the TBE1 elements. Population dynamics and evolution will be studied to distinguish between different selection models. Secondly, the developmentally regulated excision of TBE1 will be examined. Long term goals include developing an in vitro system and attempting to understand the coevolution of TBEs and their hosts.